Adjuvanted formulations of booster vaccines

ABSTRACT

The invention improves TdaP vaccines by including a TLR agonist in them. This agonist can provide stronger protection, longer-lasting protection, and/or can reduce the amount of antigen which is required to achieve a particular immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Division of co-pending U.S. patent applicationSer. No. 15/163,592, filed May 24, 2016, which is a continuation of U.S.patent application Ser. No. 13/790,948, filed Mar. 8, 2013, patented asU.S. Pat. No. 9,375,471 on Jun. 28, 2016, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application Nos. 61/608,398, filedMar. 8, 2012, and 61/697,730, filed Sep. 6, 2012, all of which arehereby expressly incorporated by reference into the present application.

TECHNICAL FIELD The invention is in the field of booster vaccines fordiphtheria, tetanus and pertussis. BACKGROUND ART

Two adolescent DTP booster vaccines are currently available—BOOSTRIX™ &ADACEL™ [1]. Both vaccines contain diphtheria toxoid, tetanus toxoid andacellular pertussis antigens. They are also available in combinationwith inactivated poliovirus (BOOSTRIX POLIO™ and ADACEL POLIO™). All ofthese vaccines include an aluminium salt adjuvant.

In general these vaccines are known as TdaP vaccines, in contrast topediatric DTaP vaccines. A common feature is that, relative to theirpediatric counterparts, they have lower antigen doses e.g. thediphtheria toxoid content of BOOSTRIX™ is 10-fold lower than in theINFANRIX™ products, and in ADACEL™ it is 7.5-fold lower than inDAPTACEL™. Moreover, the ratio of antigenic components is also altered.In particular, the ratio of diphtheria and tetanus toxoids is 2.5:1 inthe INFANRIX™ products but is 1:2 in the BOOSTRIX™ product, and is 3:1in the DAPTACEL™ product but is 1:2.5 in the ADACEL™ product. Thus thesebooster vaccines show a large reduction in the dose of diphtheriatoxoid, both in absolute amounts and also relative to the tetanus toxoidcontent. Some of the pertussis components also differ from the levelsseen in the pediatric counterparts, but the levels of the poliovirusantigens are the same in both the pediatric and adolescent vaccines.Based on public information the compositions are as follows:

D T Pa⁽¹⁾ IPV⁽³⁾ Volume Al⁺⁺⁺ Boostrix ™ 2.5 Lf 5 Lf 8/8/2.5 — 0.5 ml≤0.39 mg Boostrix 2.5 Lf 5 Lf 8/8/2.5 40/8/32 0.5 ml  0.5 mg Polio ™Adacel ™   2 Lf 5 Lf 2.5/5/3⁽²⁾ — 0.5 ml  0.33 mg Adacel   2 Lf 5 Lf2.5/5/3⁽²⁾ 40/8/32 0.5 ml  0.33 mg Polio ™ Notes: ⁽¹⁾Pa dose showsamounts of pertussis toxoid, then FHA, then pertactin (μg). ⁽²⁾Adacel'sPa components also contain 5 μg fimbriae types 2 and 3. ⁽³⁾IPV doseshows amounts of type 1, then type 2, then type 3 (measured in DU).

It is an object of the invention to provide further and improved TdaPvaccines suitable for human use as a booster in adults, adolescents andchildren aged four years and older who have previously receivedchildhood immunisation.

DISCLOSURE OF THE INVENTION

In a first aspect, the invention improves current TdaP vaccines byincluding a TLR agonist in them. This agonist can provide strongerprotection, longer-lasting protection, and/or can reduce the amount ofantigen which is required to achieve a particular immune response.

In a second aspect, the invention improves current TdaP vaccines byadjuvanting them with an oil-in-water emulsion. This emulsion can againimprove protection relative to known TdaP vaccines.

For the first aspect the invention therefore provides an immunogeniccomposition comprising a diphtheria toxoid, a tetanus toxoid, apertussis toxoid, an aluminium salt adjuvant, and a TLR agonist, whereinthe composition includes an excess of tetanus toxoid relative todiphtheria toxoid (in Lf units).

The TLR agonist is ideally a TLR4 agonist or a TLR7 agonist. Preferably,the TLR agonist and/or at least one of the toxoids is/are adsorbed tothe aluminium salt adjuvant.

By including a TLR agonist it is possible for the compositions to have alower amount of antigen and/or lower amount of aluminium relative toknown vaccines, while nevertheless having comparable immunogenicity.

Ideally the composition has one or more of the following properties:

-   -   an Al⁺⁺⁺ concentration of ≤0.5 mg/ml;    -   a diphtheria toxoid concentration of ≤4 Lf/ml;    -   a tetanus toxoid concentration of ≤9 Lf/ml; and/or    -   a pertussis toxoid concentration of ≤4 μg/ml.

For instance, where the immunogenic composition is in a unit dose formfor administration to a patient (e.g. in a 0.5 ml volume), it can haveone or more of the following properties:

-   -   an Al⁺⁺⁺ content of ≤0.255 mg;    -   a diphtheria toxoid content of ≤2 Lf;    -   a tetanus toxoid content of ≤4.5 Lf; and/or    -   a pertussis toxoid content of ≤2 μg.

For the second aspect the invention provides an immunogenic compositioncomprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid,and an oil-in-water emulsion adjuvant, wherein the composition includesan excess of tetanus toxoid relative to diphtheria toxoid (in Lf units).

Ideally the composition has one or more of the following properties:

-   -   a diphtheria toxoid concentration of ≤4 Lf/ml;    -   a tetanus toxoid concentration of ≤9 Lf/ml; and/or    -   a pertussis toxoid concentration of ≤4 μg/ml.

For instance, where the immunogenic composition is in a unit dose formfor administration to a patient (e.g. in a 0.5 ml volume), it can haveone or more of the following properties:

-   -   a diphtheria toxoid content of ≤2 Lf;    -   a tetanus toxoid content of ≤4.5 Lf; and/or    -   a pertussis toxoid content of ≤2 μg.

For both aspects, compositions of the invention can include antigens inaddition to diphtheria toxoid, tetanus toxoid, and pertussis toxoid e.g.they can include an inactivated poliovirus (IPV) component.

Aluminium Salts

TLR agonists can adsorb to insoluble aluminium salts to form an adsorbedcomplex for adjuvanting TdaP immunogens. Such aluminium salts have along history of use in vaccines.

Useful aluminium salts include, but are not limited to, aluminiumhydroxide and aluminium phosphate adjuvants. Such salts are describede.g. in chapters 8 & 9 of reference 2, and chapter 4 of reference 3.Aluminium salts which include hydroxide ions are preferred for use withthe present invention as these hydroxide ions can readily undergo ligandexchange. Thus preferred salts for adsorption of TLR agonists arealuminium hydroxide and/or aluminium hydroxyphosphate. These havesurface hydroxyl moieties which can readily undergo ligand exchange withphosphorus-containing groups (e.g. phosphates, phosphonates) to providestable adsorption. An aluminium hydroxide adjuvant is most preferred.

The adjuvants commonly known as “aluminium hydroxide” are typicallyaluminium oxyhydroxide salts, which are usually at least partiallycrystalline. Aluminium oxyhydroxide, which can be represented by theformula AlO(OH), can be distinguished from other aluminium compounds,such as aluminium hydroxide Al(OH)₃, by infrared (IR) spectroscopy, inparticular by the presence of an adsorption band at 1070cm⁻¹ and astrong shoulder at 3090-3100cm⁻¹ (chapter 9 of ref. 2). The degree ofcrystallinity of an aluminium hydroxide adjuvant is reflected by thewidth of the diffraction band at half height (WHH), withpoorly-crystalline particles showing greater line broadening due tosmaller crystallite sizes. The surface area increases as WHH increases,and adjuvants with higher WHH values have been seen to have greatercapacity for antigen adsorption. A fibrous morphology (e.g. as seen intransmission electron micrographs) is typical for aluminium hydroxideadjuvants e.g. with needle-like particles with diameters about 2 nm. ThepI of aluminium hydroxide adjuvants is typically about 11 i.e. theadjuvant itself has a positive surface charge at physiological pH.Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants commonly known as “aluminium phosphate” are typicallyaluminium hydroxyphosphates, often also containing a small amount ofsulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtainedby precipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.) indicates thepresence of structural hydroxyls (chapter 9 of reference 2).

The PO₄/Al⁺⁺⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al⁺⁺⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate-like morphology as seen intransmission electron micrographs, with primary particles in the rangeof 50 nm). Typical diameters of the particles are in the range 0.5-20 μm(e.g. about 5-10 μm) after any antigen adsorption. Adsorptive capacitiesof between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reportedfor aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

In solution both aluminium phosphate and hydroxide adjuvants tend toform stable porous aggregates 1-10 μm in diameter [4].

A composition including a TLR agonist adsorbed to an aluminium salt canalso include a buffer (e.g. a phosphate or a histidine or a Trisbuffer). When such a composition includes a phosphate buffer, however,it is preferred that the concentration of phosphate ions in the buffershould be less than 50 mM e.g. <40 mM, <30 mM, <20 mM, <10 mM, or <5 mM,or between 1-15 mM. A histidine buffer is preferred e.g. between 1-50mM, between 5-25 mM, or about 10 mM.

Because of the insolubility of adsorptive aluminium salts which areuseful with the invention, compositions containing adsorbed TLR agonistswill generally be suspensions having a cloudy appearance. This can maskcontaminating bacterial growth and so a composition of the invention mayinclude a preservative such as thiomersal or 2-phenoxyethanol. It ispreferred that a composition should be substantially free from (e.g. <10μg/ml) mercurial material e.g. thiomersal-free. Compositions containingno mercury are more preferred. Preservative-free compositions are alsopossible

A composition can include a mixture of both an aluminium hydroxide andan aluminium phosphate, and a TLR agonist may be adsorbed to one or bothof these salts.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 0.5 mg/ml e.g. ≤0.4 mg/ml, ≤0.3 mg/ml,≤0.2 mg/ml, ≤0.1 mg/ml, etc. Because the inclusion of a TLR agonist canimprove the adjuvant effect of aluminium salts then the inventionadvantageously permits lower amounts of Al⁺⁺⁺ per dose, and so acomposition of the invention can usefully include between 10 and 250 μgof Al⁺⁺ per unit dose. Current TdaP vaccines include at least 330 μgAl⁺⁺⁺ per dose. In concentration terms, a composition of the inventionmay have an Al⁺⁺⁺ concentration between 10 and 500 μg/ml e.g. between10-300 μg/ml, between 10-200 μg/ml, or between 10-100 μg/ml.

In general, the weight ratio of TLR agonist to Al⁺⁺⁺ will be less than5:1 e.g. less than 4:1, less than 3:1, less than 2:1, or less than 1:1.Thus, for example, with an Al⁺⁺⁺ concentration of 0.5 mg/ml the maximumconcentration of TLR agonist would be 2.5 mg/ml. But higher or lowerlevels can be used. A lower mass of TLR agonist than of Al⁺⁺⁺ can bemost typical e.g. per dose, 100 μg of TLR agonist with 0.2 mg Al⁺⁺⁺,etc. For instance, the Fendrix product includes 50 μg of 3d-MPL and 0.5mg Al⁺⁺⁺ per dose.

It is preferred that at least 50% (by mass) of TLR agonist(s) in thecomposition is adsorbed to the aluminium salt e.g. ≥60%, ≥70%, ≥80%,≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or even 100%.

TLR Agonists

In its first aspect, compositions of the invention include a TLR agonisti.e. a compound which can agonise a Toll-like receptor. Most preferably,a TLR agonist is an agonist of a human TLR. The TLR agonist can activateany of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11;preferably it can activate human TLR4 or human TLR7.

Agonist activity of a compound against any particular Toll-like receptorcan be determined by standard assays. Companies such as Imgenex andInvivogen supply cell lines which are stably co-transfected with humanTLR genes and NFκB, plus suitable reporter genes, for measuring TLRactivation pathways. They are designed for sensitivity, broad workingrange dynamics and can be used for high-throughput screening.Constitutive expression of one or two specific TLRs is typical in suchcell lines. See also reference 5. Many TLR agonists are known in the arte.g. reference 6 describes certain lipopeptide molecules that are TLR2agonists, references 7 to 10 each describe classes of small moleculeagonists of TLR7, and references 11 & 12 describe TLR7 and TLR8 agonistsfor treatment of diseases.

A TLR agonist used with the invention ideally includes at least oneadsorptive moiety. The inclusion of such moieties in TLR agonists allowsthem to adsorb to insoluble aluminium salts (e.g. by ligand exchange orany other suitable mechanism) and improves their immunological behaviour[13].

Phosphorus-containing adsorptive moieties are particularly useful, andso an adsorptive moiety may comprise a phosphate, a phosphonate, aphosphinate, a phosphonite, a phosphinite, etc.

Preferably the TLR agonist includes at least one phosphonate group.

Thus, in preferred embodiments, a composition of the invention includesa TLR agonist (more preferably a TLR7 agonist) which includes aphosphonate group. This phosphonate group can allow adsorption of theagonist to an insoluble aluminium salt [13].

TLR agonists useful with the invention may include a single adsorptivemoiety, or may include more than one e.g. between 2 and 15 adsorptivemoieties. Typically a compound will include 1, 2 or 3 adsorptivemoieties.

Phosphorus-containing TLR agonists useful with the invention can berepresented by formula (A1):

wherein:

-   -   R^(X) and R^(Y) are independently selected from H and C₁-C₆        alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is a linker e.g. selected from, C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6;    -   q is selected from 1, 2, 3 and 4;    -   n is selected from 1, 2 and 3; and    -   A is a TLR agonist moiety.

In one embodiment, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substitutedwith 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selectedfrom 1 and 2; and n is 1. Thus in these embodiments the adsorptivemoiety comprises a phosphate group.

In other embodiments, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is a covalent bond; L is selected fromC₁-C₆ alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)' each optionallysubstituted with 1 to 2 halogen atoms; p is selected from 1, 2 or 3; qis selected from 1 or 2; and n is 1. Thus in these embodiments theadsorptive moiety comprises a phosphonate group.

Useful ‘A’ moieties for formula (A1) include, but are not limited to,radicals of any of the following compounds, defined herein or asdisclosed in references 4-13 and 14-48:

In some embodiments, the TLR agonist moiety ‘A’ has a molecular weightof less than 1000 Da. In some embodiments, the TLR agonist of formula(A1) has a molecular weight of less than 1000 Da.

Preferred TLR agonists are water-soluble. Thus they can form ahomogenous solution when mixed in an aqueous buffer with water at pH 7at 25° C. and 1 atmosphere pressure to give a solution which has aconcentration of at least 50 μg/ml. The term “water-soluble” thusexcludes substances that are only sparingly soluble under theseconditions.

Useful TLR agonists include those having formula (C), (D), (E), (F),(G), (H), (I), (II), (J) or (K) as described in more detail below. Otheruseful TLR agonists are compounds 1 to 102 as defined in reference 13.Preferred TLR7 agonists have formula (K), such as compound K2 identifiedbelow. These can be used as salts e.g. the arginine salt of K2.

Preferred TLR4 agonists are analogs of monophosphoryl lipid A (MPL), asdescribed in more detail below. For instance, a useful TLR4 agonist is a3d-MPL.

A composition of the invention can include more than one TLR agonist.These two agonists are different from each other and they can target thesame TLR or different TLRs. Both agonists can be adsorbed to analuminium salt.

Formulae (C), (D), (E) and (H) TLR7 Agonists

The TLR agonist can be a compound according to any of formulae (C), (D),(E), and (H):

wherein:

-   -   (a) P³ is selected from H, C₁-C₆alkyl, CF₃, and        —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and        -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁴ is selected from H,        C₁-C₆alkyl, —C₁-C₆alkylaryl and -Y-L-X—P(O)(OR^(X))(OR^(Y));        with the proviso that at least one of P³ and P⁴ is        -Y-L-X—P(O)(OR^(X))(OR^(Y)),    -   (b) P⁵ is selected from H, C₁-C₆alkyl, and        -Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁶ is selected from H, C₁-C₆alkyl        each optionally substituted with 1 to 3 substituents selected        from C₁-C₄alkyl and OH, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷        is selected from H, C₁-C₆alkyl,        —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)—, —NHC₁-C₆alkyl and        -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one        of P⁵, P⁶ and P⁷ is -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   (c) P⁸ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH, and        -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are each        independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH and        C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso        that at least one of P⁸, P⁹ or P¹⁰ is        -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   (d) P¹⁶ and each P¹⁸ are each independently selected from H,        C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected        from H, C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl        heteroaryl, C₁-C₆alkylaryl-Y-L-X—P(O)(OR^(X))(OR^(Y)) and        -Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1        to 2 substituents selected from C₁-C₆alkyl or heterocyclyl with        the proviso that at least one of P¹⁶, P¹⁷ or a P¹⁸ contains a        -Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   R^(C), R^(D) and R^(H) are each independently selected from H        and C₁-C₆alkyl;    -   X^(C) is selected from CH and N;    -   R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—;    -   X^(E) is selected from a covalent bond, CR^(E2)R^(E3) and        NR^(E4);    -   R^(E2), R^(E3) and R^(E4) are independently selected from H and        C₁-C₆alkyl;    -   X^(H1)—X^(H2) is selected from —CR^(H2)R^(H3)—,        —CR^(H2)R^(H3)—CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—,        —C(O)CR^(H2)R^(H3)—, —CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—,        C(O)NR^(H4)—, CR^(H2)R^(H3)S(O)₂ and —CR^(H2)═CR^(H2)—;    -   R^(H2), R^(H3) and R^(H4) are each independently selected from        H, C₁-C₆alkyl and P¹⁸;    -   X^(H3) is selected from N and CN;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene,    -   C₁-C₆alkyleneoxy and —((CH₂)_(p)O)₉(CH₂)_(p)— each optionally        substituted with 1 to 4 substituents independently selected from        halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; m is selected        from 0 or 1;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6;    -   q is selected from 1, 2, 3 and 4; and    -   s is selected from 0 and 1.

Formula (G)—TLR8 Agonist

The TLR agonist can be a compound according to formula (G):

wherein:

-   -   P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆ alkoxy, NR^(V)R^(W)        and -Y-L-X—P(O)(OR^(C))(OR^(Y));    -   P¹² is selected from H, C₁-C₆alkyl, aryl optionally substituted        by —C(O)NR^(V)R^(W), and -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P¹³, P¹⁴ and P¹⁵ are independently selected from H, C₁-C₆alkyl,        C₁-C₆ alkoxy and -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   with the proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ or P¹⁵        is -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   R^(V) and R^(W) are independently selected from H, C₁-C₆alkyl or        together with the nitrogen atom to which they are attached form        a 4 to 7 remembered heterocyclic ring;    -   X^(G) is selected from C, CH and N;    -   represents an optional double bond, wherein X^(G) is C if        is a double bond; and    -   R^(G) is selected from H and C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6 and    -   q is selected from 1, 2, 3 and 4.        Formulae (I) and (II)—TLR 7 agonists [8]

The TLR agonist can be a compound according to formula (I) or formula(II):

wherein:

-   -   Z is —NH₂ or —OH;    -   X¹ is alkylene, substituted alkylene, alkenylene, substituted        alkenylene, alkynylene, substituted alkynylene, carbocyclylene,        substituted carbocyclylene, heterocyclylene, or substituted        heterocyclylene;    -   L¹ is a covalent bond, arylene, substituted arylene,        heterocyclylene, substituted heterocyclylene, carbocyclylene,        substituted carbocyclylene, —S—, —S(O)—, S(O)₂, —NR⁵—, or —O—    -   X² is a covalent bond, alkylene, or substituted alkylene;    -   L² is NR⁵—, —N(R⁵)C(O)—, —O—, —S—, —S(O)—, S(O)₂, or a covalent        bond;    -   R³ is H, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, heterocyclyl,        substituted heterocyclyl, heterocyclylalkyl, or substituted        heterocyclylalkyl;    -   Y¹ and Y² are each independently a covalent bond, —O— or —NR⁵—;        or —Y¹ —R¹ and —Y²—R² are each independently —O—N═C(R⁶R⁷);    -   R¹ and R² are each independently H, alkyl, substituted alkyl,        carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted        heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl,        substituted heterocyclylalkyl, -alkylene-C(O)—O—R⁵, (substituted        alkylene)-C(O)—O—R⁵, -alkylene-O—C(O)—R⁵, -(substituted        alkylene)-O—C(O)—R⁵, -alkylene-O—C(O)—O—R⁵, or -(substituted        alkylene)-O—C(O)—O—R⁵    -   R⁴ is H, halogen, —OH, —O-alkyl, —O-alkylene-O—C(O)—O—R⁵, —O—C(O        )—O—R⁵, —SH, or —NH(R⁵);    -   each R⁵, R⁶, and R⁷ are independently H, alkyl, substituted        alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl,        substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, arylalkyl, substituted arylalkyl,        heterocyclylalkyl, or substituted heterocyclylalkyl.

Formula (J)—TLR2 Agonists [14]

The TLR agonist can be a compound according to formula (J):

wherein:

-   -   R¹ is H, —C(O)—C₇-C₁₈alkyl or C(O)—C₁-C₆alkyl;    -   R² is C₇-C₁₈alkyl;    -   R³ is C₇-C₁₈alkyl;    -   L₁ is —CH₂OC(O)—, —CH₂O—, —CH₂NR⁷C(O)— or —CH₂OC(O)NR⁷—;    -   L₂ is —O—OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—;    -   R⁴ is -L₃R⁵ or -L₄R⁵;    -   R⁵ is —(R⁷)₂, —OR⁷, —P(O)(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸,        —NR⁷C(O)L₄R⁸, —OL₃R⁶, —C(O)NR⁷L₃R⁸, —C(O)NR⁷L₄R⁸, —S(O)₂OR⁷,        —OS(O)₂OR⁷, C₁-C₆alkyl, a C₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14        ring membered heteroaryl containing 1 to 3 heteroatoms selected        from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 ring membered        heterocycloalkyl containing 1 to 3 heteroatoms selected from O,        S and N, wherein the aryl, heteroaryl, cycloalkyl and        heterocycloalkyl of R⁵ are each unsubstituted or the aryl,        heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are each        substituted with 1 to 3 substituents independently selected from        —OR⁹, —OL₃R⁶, —OL₄R⁶, —OR⁷, and —C(O)OR⁷;    -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is        unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1        to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with        2 C₁-C₆alkyl groups on the same carbon atom which together,        along with the carbon atom they are attached to, form a        C₃-C₈cycloakyl;    -   L₄ is —((CR7R7)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or        —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p), wherein each R¹¹ is a        C₁-C₆alkyl groups which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   each R⁶ is independently selected from halo, C₁-C₆alkyl,        C₁-C₆alkyl substituted with 1-2 hydroxyl groups, —OR⁷, —N(R⁷)₂,        —C(O)OH, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀aryl and a        C₁₄aryl;    -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is selected from SR⁷, —C(O )OH, —P(O)(OR⁷)₂, and a 5 to 6        ring membered heterocycloalkyl containing 1 to 3 heteroatoms        selected from O and N;    -   R⁹ is phenyl;    -   each R¹⁰ is independently selected from H and halo;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

Preferably R⁵ is P(O)(OR⁷)₂, —NR⁷C(O)L₃-P(O)(OR⁷)₂,—NR⁷C(O)L₄-P(O)(OR⁷)₂, —OL₃-P(O)(OR⁷)₂, —C(O)NR⁷L₃-P(O)(OR⁷)₂, or—C(O)NR⁷L₄-P(O)(OR⁷)₂.

In some embodiments of (J), R₁ is H. In other embodiments of (J), R₁ is—C(O)—C₁₅alkyl;

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —C(O )NR⁷—; or (ii) or L₁ is —CH₂O— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —OC(O)NR⁷—; or (iii) L₁ is —CH₂NR⁷C(O)— and L₂ is —OC(O)—,—NR⁷C(O)— or —OC(O )NR⁷—; or (iv) L₁ is —CH₂OC(O)NR⁷— and L₂ is —OC(O)—,—O—, NR⁷C(O)— or —OC(O)NR⁷—.

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—; or(ii) L₁ is —CH₂O— and L₂ is —O—; or (iii) L₁ is —CH₂O— and L₂ is—NHC(O)—; or (iv) L₁ is —CH₂OC(O)NH— and L₂ is —OC(O)NH—.

In some embodiments of (J), (i) R² is —C₁₁alkyl and R³ is —C₁₁alkyl; or(ii) R² is —C₁₆alkyl and R³ is —C₁₆alkyl; or (iii) R² is —C₁₆alkyl andR³ is —C₁₁alkyl; or (iv) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (v) R²is —C₇alkyl and R³ is —C₇alkyl; or (vi) R² is —C₉alkyl and R³ is—C₉alkyl; or (vii) R² is —C₈alkyl and R³ is —C₈alkyl; or (viii) R² is—C₁₃alkyl and R³ is —C₁₃alkyl; or (ix) R² is —C₁₂alkyl and R³ is—C₁₁alkyl; or (x) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (xi) R² is—C₁₀alkyl and R³ is —C₁₀alkyl; or (xii) R² is —C₁₅alkyl and R³ is—C₁₅alkyl.

In some embodiments of (J), R² is —C₁₁alkyl and R³ is —C₁₁alkyl.

In some embodiments of (J), L₃ is a C₁-C₁₀alkylene, wherein theC₁-C₁₀alkylene of L₃ is unsubstituted or is substituted with 1 to 4 R⁶groups.

In some embodiments of (J): L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—;each R¹⁰ is independently selected from H and F; and each p isindependently selected from 2, 3, and 4.

In some embodiments of (J), each R⁶ is independently selected frommethyl, ethyl, i-propyl, i-butyl, —CH₂OH, —OH, —F, —NH₂, —C(O )OH,—C(O)NH₂, —P(O)(OH)₂ and phenyl.

In some embodiments of (J), each R⁷ is independently selected from H,methyl and ethyl.

TLR4 Agonists

Compositions of the invention can include a TLR4 agonist, and mostpreferably an agonist of human TLR4. TLR4 is expressed by cells of theinnate immune system, including conventional dendritic cells andmacrophages [15]. Triggering via TLR4 induces a signalling cascade thatutilizes both the MyD88- and TRIF-dependent pathways, leading to NF-κBand IRF3/7 activation, respectively.

TLR4 activation typically induces robust IL-12p70 production andstrongly enhances Th1-type cellular and humoral immune responses.

Various useful TLR4 agonists are known in the art, many of which areanalogs of endotoxin or lipopolysaccharide (LPS). For instance, the TLR4agonist can be:

-   -   (i) 3d-MPL (i.e. 3-O-deacylated monophosphoryl lipid A; also        known as 3-de-O-acylated monophosphoryl lipid A or        3-O-desacyl-4′-monophosphoryl lipid A). This derivative of the        monophosphoryl lipid A portion of endotoxin has a de-acylated        position 3 of the reducing end of glucosamine. It has been        prepared from a heptoseless mutant of Salmonella minnesota, and        is chemically similar to lipid A but lacks an acid-labile        phosphoryl group and a base-labile acyl group. Preparation of        3d-MPL was originally described in ref. 16, and the product has        been manufactured and sold by Corixa Corporation. It is present        in GSK's ‘AS04’ adjuvant. Further details can be found in        references 17 to 20.    -   (ii) glucopyranosyl lipid A (GLA) [21] or its ammonium salt:

-   -   (iii) an aminoalkyl glucosaminide phosphate, such as RC-529 or        CRX-524 [22-24]. RC-529 and CRX-524 have the following        structure, differing by their R₂ groups:

-   -   (iv) compounds containing lipids linked to a        phosphate-containing acyclic backbone, such as the TLR4        antagonist E5564 [25,26]:

-   -   (v) A compound of formula I, II or III as defined in reference        27, or a salt thereof, such as compounds ‘ER 803058’, ‘ER        803732’, ‘ER 804053’, ‘ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER        804680’, ‘ER 803022’, ‘ER 804764’ or ‘ER 804057’. ER 804057 is        also known as E6020 and it has the following structure:

-   -   whereas ER 803022 has the following structure:

-   -   (vi) One of the polypeptide ligands disclosed in reference 28.

Any of these TLR4 agonists can be used with the invention.

A composition of the invention can include an aluminium salt to whichthe TLR4 agonist is adsorbed. TLR4 agonists with adsorptive propertiestypically include a phosphorus-containing moiety which can undergoligand exchange with surface groups on an aluminium salt, andparticularly with a salt having surface hydroxide groups. Thus a usefulTLR4 agonist may include a phosphate, a phosphonate, a phosphinate, aphosphonite, a phosphinite, a phosphate, etc. Preferred TLR4 agonistsinclude at least one phosphate group [13] e.g. the agonists (i) to (v)listed above.

The preferred TLR4 agonist for use with the invention is 3d-MPL. Thiscan be adsorbed to an aluminium phosphate adjuvant, to an aluminiumhydroxide adjuvant, or to a mixture of both [29].

3d-MPL can take the form of a mixture of related molecules, varying bytheir acylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be ofdifferent lengths). The two glucosamine (also known as2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their2-position carbons (i.e. at positions 2 and 2′), and there is alsoO-acylation at the 3′ position. The group attached to carbon 2 hasformula —NH—CO—CH₂—CR¹R^(1′). The group attached to carbon 2′ hasformula —NH—CO—CH₂—CR²R^(2′). The group attached to carbon 3′ hasformula —O—CO—CH₂—CR³R^(3′). A representative structure is:

Groups R¹, R² and R³ are each independently (CH₂)CH₃. The value of n ispreferably between 8 and 16, more preferably between 9 and 12, and ismost preferably 10.

Groups R^(1′), R^(2′) and R^(3′) can each independently be: (a) —H; (b)—OH; or (c) —O—CO—R⁴, where R⁴ is either —H or —(CH₂)_(m)—CH₃, whereinthe value of m is preferably between 8 and 16, and is more preferably10, 12 or 14. At the 2 position, m is preferably 14. At the 2′ position,m is preferably 10. At the 3′ position, m is preferably 12. GroupsR^(1′), R^(2′) and R^(3′) are thus preferably —O-acyl groups fromdodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all of R^(1′), R^(2′) and R^(3′) are H then the 3d-MPL has only 3acyl chains (one on each of positions 2, 2′ and 3′). When only two ofR^(1′), R^(2′) and R^(3′) are H then the 3d-MPL can have 4 acyl chains.When only one of R^(1′), R^(2′) and R^(3′) is H then the 3d-MPL can have5 acyl chains. When none of R^(1′), R^(2′) and R^(3′) is H then the3d-MPL can have 6 acyl chains. The 3d-MPL used according to theinvention can be a mixture of these forms, with from 3 to 6 acyl chains,but it is preferred to include 3d-MPL with 6 acyl chains in the mixture,and in particular to ensure that the 6 acyl chain form makes up at least10% by weight of the total 3d-MPL e.g. ≥20%, ≥30%, ≥40%, ≥50% or more.3d-MPL with 6 acyl chains has been found to be the most adjuvant-activeform.

Thus the most preferred form of 3d-MPL for use with the invention is:

Where 3d-MPL is used in the form of a mixture then references to amountsor concentrations of 3d-MPL in compositions of the invention refer tothe combined 3d-MPL species in the mixture.

Typical compositions include 3d-MPL at a concentration of between 25μg/ml and 200 μg/ml e.g. in the range 50-150 μg/ml, 75-125 μg/ml, 90-110μg/ml, or about 100 μg/ml. It is usual to administer between 25-75 μg of3d-MPL per dose e.g. between 45-55 μg, or about 50 μg 3d-MPL per dose.

In aqueous conditions, 3d-MPL can form micellar aggregates or particleswith different sizes e.g. with a diameter <150 nm or >500 nm. Either orboth of these can be used with the invention, and the better particlescan be selected by routine assay. Smaller particles (e.g. small enoughto give a clear aqueous suspension of 3d-MPL) are preferred for useaccording to the invention because of their superior activity [30].Preferred particles have a mean diameter less than 150 nm, morepreferably less than 120nm, and can even have a mean diameter less than100 nm. In most cases, however, the mean diameter will not be lower than50 nm. Where 3d-MPL is adsorbed to an aluminum salt then it may not bepossible to measure the 3D-MPL particle size directly, but particle sizecan be measured before adsorption takes place. Particle diameter can beassessed by the routine technique of dynamic light scattering, whichreveals a mean particle diameter. Where a particle is said to have adiameter of x nm, there will generally be a distribution of particlesabout this mean, but at least 50% by number (e.g. ≥60%, ≥70%, ≥80%,≥90%, or more) of the particles will have a diameter within the rangex±25%.

Formula (K) [31]

The TLR agonist can be a compound according to formula (K):

wherein:

-   -   R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, -L¹R⁵, -L¹R⁶ _(, -L) ²R⁵, -L²R⁶,        or —OL²R⁶;    -   L¹ is C(O)—or —O—;    -   L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or        —((CR⁴R⁴)_(p)O)₈(CH₂)_(p), wherein the C₁-C₆alkylene and        C₂-C₆alkenylene of L² are optionally substituted with 1 to 4        fluoro groups;    -   each L³ is independently selected from C₁-C₆alkylene and        —((CR⁴R⁴)_(p)O)₈(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³ is        optionally substituted with 1 to 4 fluoro groups;    -   L⁴ is arylene or heteroarylene;    -   R² is H or C₁-C₆alkyl;    -   R³ is selected from C₁-C₄alkyl, L³R⁵, -L¹R⁵, -L³R⁷, -L³L⁴L³R⁷,        L³L⁴R⁵, -L³L⁴L³R³, —OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR⁸,        —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH;    -   each R⁴ is independently selected from H and fluoro;    -   R⁵ is —P(O)(OR⁹)₂,    -   R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;    -   R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;    -   R⁸ is H or C₁-C₄alkyl;    -   each R⁹ is independently selected from H and C₁-C₆alkyl;    -   R¹⁰ is H or C₁-C₄alkyl;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

The compound of formula (K) is preferably of formula (K′):

wherein:

-   -   P¹ is selected from H, C₁-C₆alkyl optionally substituted with        COOH and -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P² is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy and        -Y-L-X—P(O)(OR^(Xx))(OR^(Y));    -   with the proviso that at least one of P¹ and P² is        -Y-L-X—P(O)(OR^(X))(OR^(Y));    -   R^(B) is selected from H and C₁-C₆alkyl;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and    -   q is selected from 1, 2, 3 and 4.

In some embodiments of formula (K′): P¹ is selected from C₁-C₆alkyloptionally substituted with COOH and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P² isselected from C₁-C₆alkoxy and -Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(B) isC₁-C₆alkyl; X is a covalent bond; L is selected from C₁-C₆alkylene and—((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and P(O)(OH)₂; each p is independently selected from 1, 2and 3; q is selected from 1 and 2.

Formula (F)—TLR7 Agonists [9]

The TLR agonist can be a compound according to formula (F):

wherein:

-   -   X³ is N;    -   X⁴ is N or CR³    -   X⁵ is —CR⁴═CR⁵—;    -   R¹ and R² are H;    -   R³ is H;    -   R⁴ and R⁵ are each independently selected from H, halogen,        —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂,        —NHN(R⁹)₂, —SR⁷, —(CH₂),OR⁷, —(CH₂)_(n)R⁷, - LR⁸, -LR¹⁰, —OLR⁸,        —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,        C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl,        heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein        the C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,        C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,        C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ and R⁵        are each optionally substituted with 1 to 3 substituents        independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,        —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,        —P(O)(OR¹⁹)₂, —OP(O)(OR¹⁹)₂, —C(O)N(R⁹)₇, —S(O)₂R⁸, —S(O)R⁸,        —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;    -   or, R³ and R⁴, or R⁴ and R⁵, or R⁵ and R⁶, when present on        adjacent ring atoms, can optionally be linked together to form a        5-6 membered ring, wherein the 5-6 membered ring is optionally        substituted with R⁷;    -   each L is independently selected from a bond,        —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and        C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and        C₂-C₆alkynylene of L are each optionally substituted with 1 to 4        substituents independently selected from halogen, —R⁸, —OR⁸,        —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and)        —OP(O)(OR¹⁰)₂;    -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,        C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and        C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,        C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and        C₃-C₈heterocycloalkyl groups of R⁷ are each optionally        substituted with 1 to 3 R¹³ groups, and each R¹³ is        independently selected from halogen, —CN, -LR⁹, -LOR⁹, —OLR⁹,        -LR¹⁰, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸, -LSR⁸, -LSR¹⁰,        -LC(O)R⁸, —OLC(O)R⁸, -LC(O)OR⁸, -LC(O)R¹⁰, -LOC(O)OR⁸,        -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸, -LNR⁹R¹° ,        -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH, -LNR⁹C(O)R⁸,        -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂, -LNR⁹S(O)₂R⁸,        -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂, -LP(O)(OR¹⁰)₂ and        -OLP(O)(OR¹⁰)₂;    -   each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl,        C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,        C_(1I)-C₆heteroalkyl, C₃-C₈cycloalkyl, C₈heterocycloalkyl,        C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,        C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,        C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,        C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each        optionally substituted with 1 to 3 substituents independently        selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,        —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹¹, —NR¹¹R¹¹, R¹²,        —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,        —S(O)R¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and        —OP(O)(OR¹¹)₂;    -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,        —C(O)R¹¹, —C(O)OR¹¹, —S(O)₂R¹¹, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl        and C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆aIkyl        that together with N they are attached to form a        C₃-C₈heterocycloalkyl, wherein the C₃-C₈heterocycloalkyl ring        optionally contains an additional heteroatom selected from N, O        and S, and wherein the C₁-C₅ alkyl, C₁-C₆ heteroalkyl, C₃-C₆        cycloalkyl, or C₃-C₈heterocycloalkyl groups of R⁹ are each        optionally substituted with 1 to 3 substituents independently        selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, OC(O)R¹¹,        —C(O)OR¹¹, —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,        —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂ and        —OP(O)(OR¹¹)₂;    -   each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl,        C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl,        C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are        optionally substituted with 1 to 3 substituents selected from        halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸.        —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂;    -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,        C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the        C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R¹¹ and R¹²        are each optionally substituted with 1 to 3 substituents        independently selected from halogen, —CN, R⁸, —OR⁸, C(O)R⁸,        OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸,        —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸, —S(O)₂N(R⁹)₂,        —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and C₁-C₆haloalkoxy;    -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken        together with the N atom to which they are attached form an        optionally substituted C₃-C₈heterocycloalkyl ring optionally        containing an additional heteroatom selected from N, O and S;    -   ring A is an aryl or a heteroaryl, wherein the aryl and        heteroaryl groups of Ring A are optionally substituted with 1 to        3 R^(A) groups, wherein each R^(A) is independently selected        from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN,        —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂,        —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸,        —C(O)C(O)R⁸, —C(O)CH2C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸,        —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,        —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)2R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,        —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,        —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸, —CH═CHCO₂R⁸,        —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸ or two adjacent R^(A)        substituents on Ring A form a 5-6 membered ring that contains up        to two heteroatoms as ring members;    -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7        or 8;    -   each m is independently selected from 1, 2, 3, 4, 5 and 6, and    -   t is 1, 2, 3, 4, 5, 6, 7 or 8.

Formulae (C), (D), (E), (G) and (H)

As discussed above, the TLR agonist can be of formula (C), (D), (E), (G)or (H).

The ‘parent’ compounds of formulae (C), (D), (E) and (H) are useful TLR7agonists (see references 7-10 and 32-48) but are preferably modifiedherein by attachment of a phosphorus-containing moiety.

In some embodiments of formulae (C), (D) and (E) the compounds havestructures according to formulae (C′), (D′) and (E′), shown below:

The embodiments of the invention of formulae (C), (D), (E) and (H) alsoapply to formulae (C′), (D′), (E′) and (H′).

In some embodiments of formulae (C), (D), (E), and (H): X is O; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl,CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and-Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and-Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C) is CH; X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; q is 1 or 2.

In other embodiments of formulae (C), (D), (E), and (H): X is a covalentbond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)—each optionally substituted with 1 to 4 substituents independentlyselected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl,CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and-Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and-Y-L-X—-P(O)(OR^(X))(OR^(Y)); X^(C) is N; X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; q is selected from 1 and 2.

In other embodiments of formula (D): P⁵ is selected from C₁-C₆alkyl, and-Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (D): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (D): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is O; L is selected fromC₁-C₆alkylene and —((CH2)_(p)O)_(q)(CH2)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)2 and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X^(E) is CH₂, P⁸ is C₁-C₆alkoxyoptionally substituted with -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (E): P⁹ is —NHC₁-C₆alkyl optionallysubstituted with OH and C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, a compound of formula (C) is not a compound inwhich P⁴ is -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, in a compound of formula (C), P⁴ is selected fromH, C₁-C₆alkyl, —C₁-C₆alkylaryl.

In some embodiments of formula (H): X^(H1)—X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H, X^(H3) is N, X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (H): X^(H1)—X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H, X^(H3) is N, X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)2 and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

The ‘parent’ compounds of formula (G) are useful TLR8 agonists (seereferences 11 & 12) but are preferably modified herein by attachment ofa phosphorus-containing moiety to permit adsorption. In some embodimentsof formula (G), the compounds have structures according to formula (G′);

In some embodiments of formula (G) or (G′): X^(G) is C and

represents a double bond.

In some embodiments of formula (G) or (G′): X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (G) or (G′): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

Oil-in-Water Emulsion Adjuvants

According to the invention's second aspect a vaccine is adjuvanted withan oil-in-water emulsion. Various such emulsions are known e.g. MF59 andAS03 are both authorised in Europe.

Useful emulsion adjuvants they typically include at least one oil and atleast one surfactant, with the oil(s) and surfactant(s) beingbiodegradable (metabolisable) and biocompatible. The oil droplets in theemulsion generally have a sub-micron diameter, and these small sizes canreadily be achieved with a microfluidiser to provide stable emulsions,or by alternative methods e.g. phase inversion. Emulsions in which atleast 80% (by number) of droplets have a diameter of less than 220 nmare preferred, as they can be subjected to filter sterilization.

The emulsion can include oil(s) from an animal (such as fish) and/orvegetable source. Sources for vegetable oils include nuts, seeds andgrains. Peanut oil, soybean oil, coconut oil, and olive oil, the mostcommonly available, exemplify the nut oils. Jojoba oil can be used e.g.obtained from the jojoba bean. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used. 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolisable and may therefore be used with theinvention. The procedures for separation, purification, saponificationand other means necessary for obtaining pure oils from animal sourcesare well known in the art.

Most fish contain metabolisable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Shark liver oil contains a branched, unsaturated terpenoids known assqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene,which is particularly preferred for use with the invention (see below).Squalane, the saturated analog to squalene, is also a useful oil. Fishoils, including squalene and squalane, are readily available fromcommercial sources or may be obtained by methods known in the art. Otherpreferred oils are the tocopherols (see below). Mixtures of oils can beused.

Preferred amounts of total oil (% by volume) in an adjuvant emulsion arebetween 1 and 20% e.g. between 2-10%. A squalene content of 5% by volumeis particularly useful.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10 e.g. about 15. The invention can be used with surfactants including,but not limited to: the polyoxyethylene sorbitan esters surfactants(commonly referred to as the Tweens), especially polysorbate 20 orpolysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO),and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such aslinear EO/PO block copolymers; octoxynols, which can vary in the numberof repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9(Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particularinterest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);phospholipids such as phosphatidylcholine (lecithin); nonylphenolethoxylates, such as the Tergitol™ NP series; polyoxyethylene fattyethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known asBrij surfactants), such as triethyleneglycol monolauryl ether (Brij 30);and sorbitan esters (commonly known as the Spans), such as sorbitantrioleate (Span 85) or sorbitan monolaurate.

Emulsions used with the invention preferably include non-ionicsurfactant(s). Preferred surfactants for including in the emulsion arepolysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80), Span 85(sorbitan trioleate), lecithin or Triton X-100. Mixtures of surfactantscan be used e.g. a mixture of polysorbate 80 and sorbitan trioleate. Acombination of a polyoxyethylene sorbitan ester such as polysorbate 80(Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol(Triton X-100) is also useful . Another useful combination compriseslaureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.Where a mixture of surfactants is used then the HLB of the mixture iscalculated according to their relative weightings (by volume) e.g. thepreferred 1:1 mixture by volume of polysorbate 80 and sorbitan trioleatehas a HLB of 8.4.

Preferred amounts of total surfactant (% by volume) in an adjuvantemulsion are between 0.1 and 2% e.g. between 0.25-2%. A total content of1% by volume is particularly useful e.g. 0.5% by volume of polysorbate80 and 0.5% by volume of sorbitan trioleate.

Useful emulsions can be prepared using known techniques e.g. seereferences 3 and 49-55.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, polysorbate 80, and sorbitan        trioleate. The composition of the emulsion by volume can be        about 5% squalene, about 0.5% polysorbate 80 and about 0.5%        sorbitan trioleate. In weight terms, these ratios become 4.3%        squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This        adjuvant is known as ‘MF59’ [54-56], as described in more detail        in Chapter 10 of ref. 2 and chapter 12 of ref. 3. The MF59        emulsion advantageously includes citrate ions e.g. 10 mM sodium        citrate buffer.    -   An emulsion of squalene, a tocopherol, and polysorbate 80. The        emulsion may include phosphate buffered saline. These emulsions        may have from 2 to 10% squalene, from 2 to 10% tocopherol and        from 0.3 to 3% polysorbate 80, and the weight ratio of        squalene:tocopherol is preferably ≤1 (e.g. 0.90) as this can        provide a more stable emulsion. Squalene and polysorbate 80 may        be present volume ratio of about 5:2, or at a weight ratio of        about 11:5. Thus the three components (squalene, tocopherol,        polysorbate 80) may be present at a weight ratio of        1068:1186:485 or around 55:61:25. This adjuvant is known as        ‘AS03’. Another useful emulsion of this type may comprise, per        human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4        mg polysorbate 80 [57] e.g. in the ratios discussed above.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [58].    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 59, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [60]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. It may also        include a TLR4 agonist, such as one whose chemical structure        does not include a sugar ring [61]. Such emulsions may be        lyophilized. The ‘AF03’ product is one such emulsion.

Preferred oil-in-water emulsions used with the invention comprisesqualene and polysorbate 80.

The emulsions may be mixed with TdaP antigens during vaccinemanufacture, or they may be mixed extemporaneously at the time ofdelivery. Thus, in some embodiments, the adjuvant and antigens may bekept separately in a packaged or distributed vaccine, ready for finalformulation at the time of use. At the time of mixing (whether duringbulk manufacture, or at the point of use) the antigen will generally bein an aqueous form, such that the final vaccine is prepared by mixingtwo liquids. The volume ratio of the two liquids for mixing can vary(e.g. between 5:1 and 1:5) but is generally about 1:1. If emulsion andantigen are stored separately in a kit then the product may be presentedas a vial containing emulsion and a vial containing aqueous antigen, formixing to give adjuvanted liquid vaccine (monodose or multi-dose).

Preferred emulsions of the invention include squalene oil. This isusually prepared from shark oil but alternative sources are known e.g.see references 62 (yeast) and 63 (olive oil). Squalene which containsless than 661 picograms of PCBs per gram of squalene (TEQ) is preferredfor use with the invention, as disclosed in reference 64. The emulsionsare preferably made from squalene of high purity e.g. prepared bydouble-distillation as disclosed in reference 65.

Where a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξtocopherols can be used, but α-tocopherols are preferred. The tocopherolcan take several forms e.g. different salts and/or isomers. Saltsinclude organic salts, such as succinate, acetate, nicotinate, etc.D-α-tocopherol and DL-α-tocopherol can both be used. Tocopherols haveantioxidant properties that may help to stabilize the emulsions [66]. Apreferred α-tocopherol is DL-α-tocopherol, and a preferred salt of thistocopherol is the succinate.

Diphtheria Toxoid

Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positivenon-sporing aerobic bacterium. This organism expresses aprophage-encoded ADP-ribosylating exotoxin (‘diphtheria toxin’), whichcan be treated (e.g. using formaldehyde) to give a toxoid that is nolonger toxic but that remains antigenic and is able to stimulate theproduction of specific anti-toxin antibodies after injection. Diphtheriatoxoids are disclosed in more detail in chapter 13 of reference 67.Preferred diphtheria toxoids are those prepared by formaldehydetreatment. The diphtheria toxoid can be obtained by growing C.diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fentonmedium), which may be supplemented with bovine extract, followed byformaldehyde treatment, ultrafiltration and precipitation. The toxoidedmaterial may then be treated by a process comprising sterile filtrationand/or dialysis.

Quantities of diphtheria toxoid can be expressed in international units(IU). For example, the NIBSC [68] supplies the ‘Diphtheria ToxoidAdsorbed Third International Standard 1999’ [69,70], which contains 160IU per ampoule. As an alternative to the IU system, the ‘Lf’ unit(“flocculating units”, the “limes flocculating dose”, or the “limit offlocculation”) is defined as the amount of toxoid which, when mixed withone International Unit of antitoxin, produces an optimally flocculatingmixture [71]. For example, the NIBSC supplies ‘Diphtheria Toxoid, Plain’[72], which contains 300 Lf per ampoule and ‘The 1st InternationalReference Reagent For Diphtheria Toxoid For Flocculation Test’ [73]which contains 900 Lf per ampoule. The concentration of diphtheria toxinin a composition can readily be determined using a flocculation assay bycomparison with a reference material calibrated against such referencereagents. The conversion between IU and Lf systems depends on theparticular toxoid preparation.

The concentration of diphtheria toxoid in a composition of the inventionis typically in the range of 2-8 Lf/ml (e.g. 4 Lf/ml or 5 Lf/ml), but isideally ≤4 Lf/ml. In a typical 0.5 ml unit dose volume, therefore, theamount of diphtheria toxoid can be 2 Lf, 2.5 Lf, or less than 2 Lf.

Diphtheria toxoid in the composition is preferably adsorbed (morepreferably totally adsorbed) onto an aluminium salt, preferably onto analuminium hydroxide adjuvant.

Tetanus Toxoid

Tetanus is caused by Clostridium tetani, a Gram-positive, spore-formingbacillus. This organism expresses an endopeptidase (‘tetanus toxin’),which can be treated to give a toxoid that is no longer toxic but thatremains antigenic and is able to stimulate the production of specificanti-toxin antibodies after injection. Tetanus toxoids are disclosed inmore detail in chapter 27 of reference 67. Preferred tetanus toxoids arethose prepared by formaldehyde treatment. The tetanus toxoid can beobtained by growing C. tetani in growth medium (e.g. a Latham mediumderived from bovine casein), followed by formaldehyde treatment,ultrafiltration and precipitation. The material may then be treated by aprocess comprising sterile filtration and/or dialysis.

Quantities of tetanus toxoid can be expressed in international units(IU). For example, NIBSC supplies the ‘Tetanus Toxoid Adsorbed ThirdInternational Standard 2000’ [74,75], which contains 469 IU per ampoule.As with diphtheria toxoid, the ‘Lf’ unit is an alternative to the IUsystem. NIBSC supplies ‘The 1st International Reference Reagent forTetanus Toxoid For Flocculation Test’ [76] which contains 1000 LF perampoule. The concentration of diphtheria toxin in a composition canreadily be determined using a flocculation assay by comparison with areference material calibrated against such reference reagents.

The concentration of tetanus toxoid in a composition of the invention istypically in the range of 5-15 Lf/ml (e.g. 10 Lf/ml), but is ideally ≤9Lf/ml. In a typical 0.5 ml unit dose volume, therefore, the amount ofdiphtheria toxoid can be 5 Lf, or less than 4.5 Lf.

Tetanus toxoid in the composition is preferably adsorbed (sometimestotally adsorbed) onto that salt, preferably onto an aluminium hydroxideadjuvant.

Pertussis Toxoid

Bordetella pertussis causes whooping cough. Pertussis antigens invaccines are either cellular (whole cell, in the form of inactivated B.pertussis cells; ‘wP’) or acellular ('aP'). Preparation of cellularpertussis antigens is well documented (e.g. see chapter 21 of reference67) e.g. it may be obtained by heat inactivation of phase I culture ofB. pertussis. The invention preferably uses acellular antigens,including, one, two or (preferably) three of the following threeantigens: (1) detoxified pertussis toxin (pertussis toxoid, or ‘PT’);(2) filamentous hemagglutinin (‘FHA’); (3) pertactin (also known as the‘69 kiloDalton outer membrane protein’). These three antigens can beprepared by isolation from B. pertussis culture grown in modifiedStainer-Scholte liquid medium. PT and FHA can be isolated from thefermentation broth (e.g. by adsorption on hydroxyapatite gel), whereaspertactin can be extracted from the cells by heat treatment andflocculation (e.g. using barium chloride). The antigens can be purifiedin successive chromatographic and/or precipitation steps. PT and FHA canbe purified by hydrophobic chromatography, affinity chromatography andsize exclusion chromatography. Pertactin can be purified by ion exchangechromatography, hydrophobic chromatography and size exclusionchromatography, or by IMAC. FHA and pertactin may be treated withformaldehyde prior to use according to the invention. PT is preferablydetoxified by treatment with formaldehyde and/or glutaraldehyde. As analternative to this chemical detoxification procedure the PT may be amutant PT in which enzymatic activity has been reduced by mutagenesis[77] (e.g. the 9K/129G double mutant [78]).

The invention preferably uses a PT-containing aP antigen. When using anaP antigen a composition of the invention will typically, in addition tothe PT, include FHA and, optionally, pertactin. It can also optionallyinclude fimbriae types 2 and 3.

Quantities of acellular pertussis antigens are typically expressed inmicrograms. The concentration of pertussis toxoid in a composition ofthe invention is typically in the range of 2-20 μg/ml (e.g. 5 μg/ml or16 μg/ml), but is ideally ≤4 μg/ml. In a typical 0.5 ml unit dosevolume, therefore, the amount of diphtheria toxoid can be 2.5 μg, 8 μg,or <2 μg.

It is usual that each of pertussis toxoid, FHA and pertactin are presentin a composition of the invention. These may be present at variousratios (by mass), such as PT:FHA:p69 ratios of 16:16:5 or 5:10:6. Eachof these three antigens will generally be present at <20 μg/ml e.g. eachin the range of 4-20 μg/ml. A total pertussis antigen concentration of<40 μg/ml is typical. It is usual to have a mass excess of FHA relativeto pertactin.

Pertussis toxoid in the composition is preferably adsorbed (sometimestotally adsorbed) onto that salt, preferably onto an aluminium hydroxideadjuvant. Any FHA can also be adsorbed to an aluminium hydroxideadjuvant. Any pertactin can be adsorbed to an aluminium phosphateadjuvant.

Inactivated Poliovirus Antigen (IPV)

Poliomyelitis can be caused by one of three types of poliovirus. Thethree types are similar and cause identical symptoms, but they areantigenically very different and infection by one type does not protectagainst infection by others. As explained in chapter 24 of reference 67,it is therefore preferred to use three poliovirus antigens with theinvention—poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2(e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain). As analternative to these strains (“Salk” strains), Sabin strains of types 1to 3 can be used e.g. as discussed in references 79 & 80. These strainscan be more potent than the normal Salk strains.

Polioviruses may be grown in cell culture. A preferred culture uses aVero cell line, which is a continuous cell line derived from monkeykidney. Vero cells can conveniently be cultured microcarriers. Cultureof the Vero cells before and during viral infection may involve the useof bovine-derived material, such as calf serum, and of lactalbuminhydrolysate (e.g. obtained by enzymatic degradation of lactalbumin).Such bovine-derived material should be obtained from sources which arefree from BSE or other TSEs. The final vaccine preferably contains lessthan 10 ng/ml, preferably ≤1 ng/ml e.g. ≤500 pg/ml or ≤50 pg/ml of Verocell DNA e.g. less than 10 ng/ml of Vero cell DNA that is ≥50 base pairslong.

After growth, virions may be purified using techniques such asultrafiltration, diafiltration, and chromatography. Prior toadministration to patients, polioviruses must be inactivated, and thiscan be achieved by treatment with formaldehyde before the viruses areused in the process of the invention.

The viruses are preferably grown, purified and inactivated individually,and are then combined to give a bulk mixture for use with the invention.

Quantities of inactivated poliovirus (IPV) are typically expressed inthe ‘DU’ unit (the “D-antigen unit” [81]). Typically a composition hasIPV antigens 1/2/3 at concentrations of 80/16/64 DU/ml (40/8/32 DU per0.5 ml dose). In some embodiments, however, a composition can includelower amounts of poliovirus antigens, either through including lessantigen or by using more potent strains. For a Type 1 poliovirus theconcentration of the virus in the composition can be ≤20 DU/ml e.g. <18,<16, <14, <12, <10, etc. For a Type 2 poliovirus the concentration ofthe virus in the composition can be ≤4 DU/ml e.g. <3, <2, <1, <0.5, etc.For a Type 3 poliovirus the concentration of the virus in thecomposition can be ≤16 DU/ml e.g. <14, <12, <10, <8, <6, etc. Where allthree of Types 1, 2 and 3 poliovirus are present the three antigens canbe present at a DU ratio of 5:1:4 respectively, or at any other suitableratio e.g. a ratio of 15:32:45 when using Sabin strains [79]. A low doseof antigen from Sabin strains is particularly useful, with ≤10 DU type1, ≤20 DU type 2, and ≤30 DU type 3 (per unit dose).

Polioviruses are preferably not adsorbed to any adjuvant before they areformulated, but after formulation they may become adsorbed ontoaluminium salt(s) in the composition.

Combination Vaccines

As well as including D, T, Pa, and/or poliovirus antigens, immunogeniccompositions of the invention may include antigens from furtherpathogens. For example, these antigens may be HBsAg, conjugated Hibcapsular saccharide, conjugated N.meningitidis capsular saccharide (oneor more of serogroups A, C, W135 and/or Y) or conjugated S.pnetimonidecapsular saccharide. For example, any of the suitable antigen componentsof PEDIARIX, MENVEO, MENACTRA, NIMENRIX, PREVNAR, or SYNFLORIX can beused.

Preferably, however, the vaccine's only antigenic components are either(i) for diphtheria, tetanus and pertussis or (ii) for diphtheria,tetanus, pertussis and poliovirus.

Immunogenic compositions of the invention include at least a diphtheriatoxoid, a tetanus toxoid, and a pertussis toxoid as immunogeniccomponents. The compositions include an excess of tetanus toxoidrelative to diphtheria toxoid. This excess is measured in Lf units (butthe excess can also be seen via IU e.g. Adacel's content is quoted as 5Lf of tetanus toxoid and 2 Lf of diphtheria toxoid, or as 20IU oftetanus toxoid and 2 IU of diphtheria toxoid). The excess is ideally atleast 1.5:1 (i.e. at least 1.5 Lf of tetanus toxoid for every 1 Lf ofdiphtheria toxoid) e.g. 2:1 or 2.5:1. The excess will not usually bemore than 5-fold (again, in Lf units).

As an independent embodiment of the disclosure, the invention providesan immunogenic composition comprising an acellular pertussis componentin which a pertussis toxoid (e.g. the PT-9K/129G double mutant),filamentous hemagglutinin and pertactin are present at a mass ratio of1:1:2. For instance, the composition can comprise 4 μg pertussis toxoid,4 μg FHA and 8 μg pertactin per unit dose. The composition can alsoinclude diphtheria toxoid (e.g. 2 Lf per unit dose) and tetanus toxoid(e.g. 5 Lf per dose) to provide a TdaP combination. The composition canalso include an adjuvant comprising an aluminium salt (e.g. an aluminiumhydroxide) and, optionally, a TLR agonist as described elsewhere herein.

Pharmaceutical Compositions and Products

The invention provides various immunogenic compositions. These areideally pharmaceutical compositions suitable for use in humans.Pharmaceutical compositions usually include components in addition tothe immunogen and the adjuvant components e.g. they typically includeone or more pharmaceutical carrier(s) and/or excipient(s). A thoroughdiscussion of such components is available in reference 82.

Pharmaceutical compositions are preferably in aqueous form, particularlyat the point of administration, but they can also be presented innon-aqueous liquid forms or in dried forms e.g. as gelatin capsules, oras lyophilisates, etc.

Pharmaceutical compositions may include one or more preservatives, suchas thiomersal or 2-phenoxyethanol. Mercury-free compositions arepreferred, and preservative-free vaccines can be prepared.Pharmaceutical compositions can include a physiological salt, such as asodium salt e.g. to control tonicity. Sodium chloride (NaCl) is typical,which may be present at between 1 and 20 mg/ml e.g. 10±2 mg/ml or 9mg/ml. Other salts that may be present include potassium chloride,potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesiumchloride, calcium chloride, etc.

Pharmaceutical compositions can have an osmolality of between 200mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between290-310 mOsm/kg. Compositions may be isotonic with humans.

Pharmaceutical compositions may include compounds (with or without aninsoluble aluminium salt) in plain water (e.g. w.f.i.) but will usuallyinclude one or more buffers. Typical buffers include: a phosphate buffer(except in the fifteenth aspect); a Tris buffer; a borate buffer; asuccinate buffer; a histidine buffer (particularly with an aluminiumhydroxide adjuvant); or a citrate buffer. Buffer salt s will typicallybe included in the 5-20mM range. If a phosphate buffer is used then theconcentration of phosphate ions should, in some embodiments, be <50 mM(see above) e.g. <10 mM.

The pH of a composition of the invention will generally be between 6.0and 7.5. A manufacturing process may therefore include a step ofadjusting the pH of a composition prior to packaging. Aqueouscompositions administered to a patient can have a pH of between 5.0 and7.5, and more typically between 5.0 and 6.0 for optimum stability; wherea diphtheria toxoid and/or tetanus toxoid is present, the pH is ideallybetween 6.0 and 7.0.

Pharmaceutical compositions are preferably sterile.

Pharmaceutical compositions preferably non-pyrogenic e.g. containing <1EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EUper dose. 1 EU is equal to 0.2 ng FDA reference standard Endotoxin EC-2‘RSE’) per dose.

Pharmaceutical compositions are preferably gluten free.

During manufacture, dilution of components to give desired finalconcentrations will usually be performed with WFI (water for injection).

Pharmaceutical compositions are suitable for administration to animal(and, in particular, human) patients, and thus include both human andveterinary uses. They may be used in a method of raising an immuneresponse in a patient, comprising the step of administering thecomposition to the patient.

The invention can provide bulk material which is suitable for packaginginto individual doses, which can then be distributed for administrationto patients. Concentrations discussed above are typically concentrationsin final packaged dose, and so concentrations in bulk vaccine may behigher (e.g. to be reduced to final concentrations by dilution).

Pharmaceutical compositions may be prepared in unit dose form. In someembodiments a unit dose may have a volume of between 0.05-1.5 ml e.g.about 0.5 ml for intramuscular injection. References to 0.5 ml doseswill be understood to include normal variance e.g. 0.5 ml±0.05 ml. Formultidose situations, multiple dose amounts will be extracted andpackaged together in a single container e.g. 5 ml for a 10-dosemultidose container (or 5.5 ml with 10% overfill).

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention e.g. containing a unit dose. This devicecan be used to administer the composition to a vertebrate subject.

The invention also provides a sterile container (e.g. a vial) containinga pharmaceutical composition of the invention e.g. containing a unitdose.

The invention also provides a unit dose of a pharmaceutical compositionof the invention.

The invention also provides a hermetically sealed container containing apharmaceutical composition of the invention. Suitable containers includee.g. a vial.

Pharmaceutical compositions of the invention may be prepared in variousforms. For example, the compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared (e.g. a lyophilised composition or a spray-freeze driedcomposition), although aqueous compositions are preferred. Suspensionsfor intramuscular or intradermal or subcutaneous injection are typical.

Compositions of the first aspect comprise an effective amount of a TLRagonist i.e. an amount which, when administered to an individual, eitherin a single dose or as part of a series, is effective for enhancing theimmune response to a its co-administered immunogens. This amount canvary depending upon the health and physical condition of the individualto be treated, age, the taxonomic group of individual to be treated(e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors.

The amount of TLR agonist in a unit dose of a composition will fall in arelatively broad range that can be determined through routine trials. Anamount of between 1-1000 μg/dose can be used e.g. from 5-100 μg per doseor from 10-100 μg per dose, and ideally ≤300 μg per dose e.g. about 5μg, 10 μg, 20 μg, 25 μg, 50 μg or 100 μg per dose. Thus theconcentration of a TLR agonist in a composition of the invention may befrom 2-2000 μg/ml e.g. from 10-200 μg/ml, or about 5, 10, 20, 40, 50,100 or 200 μg/ml, and ideally ≤600 μg/ml.

Methods of Treatment, and Administration of Immunogenic Compositions

The invention is suitable for raising immune responses in humans, butthey may also be useful in non-human animals (in particular mammals)subjects. Compositions prepared according to the invention may be usedto treat both children and adults.

The invention provides a method of raising an immune response in asubject, comprising the step of administering to the subject acomposition of the invention. The invention also provides a compositionof the invention, for use in a method of raising an immune response in asubject. The invention also provides the use of (i) a TLR agonist asdefined herein and (ii) an insoluble aluminium salt and (iii) at leastdiphtheria, tetanus and pertussis toxoids, in the manufacture of amedicament (e.g. a vaccine) for raising an immune response in a subject.The invention also provides the use of (i) an oil-in-water emulsionadjuvant as defined herein and (ii) at least diphtheria, tetanus andpertussis toxoids, in the manufacture of a medicament (e.g. a vaccine)for raising an immune response in a subject.

The immune response stimulated by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.The immune response can also include a cellular response. Methods forassessing antibody and cellular immune responses after immunisation arewell known in the art, particularly for diphtheria, tetanus, pertussisand poliovirus antigens.

Administration of compositions of the invention will generally be byinjection, and this may be by the subcutaneous, intradermal of theintramuscular route. Intramuscular injection is preferred.

Immunogenic compositions of the invention will generally be administeredto people at least 3 years of age, and preferably at least 4 years ofage. For instance, the subject may be 10-64 years old, or a teenager.The compositions are most useful in people who have previously receivedroutine childhood immunisations (including DTP vaccine).

A patient will generally receive the composition as frequently asrequired. Each course of immunisation will involve a single boosterdose.

Chemical Groups Unless specifically defined elsewhere, the chemicalgroups discussed herein have the following meaning when used in presentspecification:

The term “alkyl” includes saturated hydrocarbon residues including:

-   -   linear groups up to 10 atoms (C₁-C₁₀), or of up to 6 atoms        (C₁-C₆), or of up to 4 atoms (C₁-C₄). Examples of such alkyl        groups include, but are not limited, to C₁-methyl, C₂-ethyl,        C₃-propyl and C₄-n-butyl.    -   branched groups of between 3 and 10 atoms (C₃-C₁₀), or of up to        7 atoms (C₃-C₇), or of up to 4 atoms (C₃-C₄). Examples of such        alkyl groups include, but are not limited to, C₃-iso-propyl,        C₄-sec-butyl, C₄-iso-butyl, C₄-tert-butyl and C₅-neo-pentyl.

The term “alkylene” refers to the divalent hydrocarbon radical derivedfrom an alkyl group, and shall be construed in accordance with thedefinition above.

The term “alkenyl” includes monounsaturated hydrocarbon residuesincluding:

-   -   linear groups of between 2 and 6 atoms (C2-C₆). Examples of such        alkenyl groups include, but are not limited to, C₂-vinyl,        C₃-1-propenyl, C₃-allyl, C₄-2-butenyl    -   branched groups of between 3 and 8 atoms (C₃-C₈). Examples of        such alkenyl groups include, but are not limited to,        C₄-2-methyl-2-propenyl and C₆-2,3-dimethyl-2-butenyl.

The term alkenylene refers to the divalent hydrocarbon radical derivedfrom an alkenyl group, and shall be construed in accordance with thedefinition above.

The term “alkoxy” includes 0-linked hydrocarbon residues including:

-   -   linear groups of between 1 and 6 atoms (C₁-C₆), or of between 1        and 4 atoms (C₁-C₄). Examples of such alkoxy groups include, but        are not limited to, C₁-methoxy, C₂-ethoxy, C₃-n-propoxy and        C₄-n-butoxy.    -   branched groups of between 3 and 6 atoms (C₃-C₆) or of between 3        and 4 atoms (C₃-C₄).

Examples of such alkoxy groups include, but are not limited to,C₃-iso-propoxy, and C₄-sec-butoxy and tert-butoxy.

Halo is selected from Cl, F, Br and I. Halo is preferably F.

The term “aryl” includes a single or fused aromatic ring systemcontaining 6 or 10 carbon atoms; wherein, unless otherwise stated, eachoccurrence of aryl may be optionally substituted with up to 5substituents independently selected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy,OH, halo, CN, COOR¹⁴, CF₃ and NR¹⁴R¹⁵; as defined above. Typically, arylwill be optionally substituted with 1, 2 or 3 substituents. Optionalsubstituents are selected from those stated above. Examples of suitablearyl groups include phenyl and naphthyl (each optionally substituted asstated above). Arylene refers the divalent radical derived from an arylgroup, and shall be construed in accordance with the definition above.

The term “heteroaryl” includes a 5, 6, 9 or 10 membered mono- orbi-cyclic aromatic ring, containing 1 or 2 N atoms and, optionally, anNR¹⁴ atom, or one NR¹⁴ atom and an S or an O atom, or one S atom, or oneO atom; wherein, unless otherwise stated, said heteroaryl may beoptionally substituted with 1, 2 or 3 substituents independentlyselected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, halo, CN, COOR¹⁴, CF₃ andNeR¹⁵; as defined below. Examples of suitable heteroaryl groups includethienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl,tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl,benzimidazolyl, benzotriazolyl, quinolinyl and isoquinolinyl (optionallysubstituted as stated above).

Heteroarylene refers the divalent radical derived from heteroaryl, andshall be construed in accordance with the definition above.

The term “heterocyclyl” is a C-linked or N-linked 3 to 10 memberednon-aromatic, mono- or bi-cyclic ring, wherein said heterocycloalkylring contains, where possible, 1, 2 or 3 heteroatoms independentlyselected from N, NR¹⁴, S(O)_(q) and O; and said heterocycloalkyl ringoptionally contains, where possible, 1 or 2 double bonds, and isoptionally substituted on carbon with 1 or 2 substituents independentlyselected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, CN, CF₃, halo, COOR¹⁴,NR¹⁴R¹⁵ and aryl.

In the above definitions R¹⁴ and R¹⁵ are independently selected from Hand (C₁-C₆)alkyl.

When a structural formula is defined with a substituent attached to thecore of the molecule by an unspecified, or “floating” bond, for example,as for the group P³ in the case of formula (C), this definitionencompasses the cases where the unspecified substituent is attached toany of the atoms on the ring in which the floating bond is located,whilst complying with the allowable valence for that atom.

In the case of compounds of the invention which may exist in tautomericforms (i.e. in keto or enol forms), for example the compounds of formula(C) or (H), reference to a particular compound optionally includes allsuch tautomeric forms.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X +Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

As animal (and particularly bovine) materials are typically used in theculture of cells, they should be obtained from sources that are freefrom transmissible spongiform encaphalopathies (TSEs), and in particularfree from bovine spongiform encephalopathy (BSE).

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Phosphorous-containing groups employed with the invention may exist in anumber of protonated and deprotonated forms depending on the pH of thesurrounding environment, for example the pH of the solvent in which theyare dissolved. Therefore, although a particular form may be illustratedit is intended, unless otherwise mentioned, for these illustrations tomerely be representative and not limiting to a specific protonated ordeprotonated form. For example, in the case of a phosphate group, thishas been illustrated as OP(O)(OH)₂ but the definition includes theprotonated forms —[OP(O)(OH₂)(OH)]⁺ and —[OP(O)(OH₂)₂]²⁺ that may existin acidic conditions and the deprotonated forms —[OP(O)(OH)(O)]⁻ and[OP(O)(O)₂]²⁻ that may exist in basic conditions.

Compounds disclosed herein can exist as pharmaceutically acceptablesalts. Thus, the compounds may be used in the form of theirpharmaceutically acceptable salts i.e. physiologically ortoxicologically tolerable salt (which includes, when appropriate,pharmaceutically acceptable base addition salts and pharmaceuticallyacceptable acid addition salts).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the % of FHA-specific memory B cells for the indicatedtreatment groups.

MODES FOR CARRYING OUT THE INVENTION Vaccine Preparation

References 31 and 83 disclose TLR7 agonists having formula (K) asdiscussed above. One of these compounds,3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f]-[1,7]naphthyridin-8-yl)propanoic acid is referred to hereafter ascompound “K2”:

Compound K2 is added to water at 4mg/ml, then 1M NaOH is added to ensurefull solubilisation, with stirring for 15 minutes at room temperature.This material is added to a suspension of aluminium hydroxide adjuvant(Al—H) to give the desired final concentration. This mixture is shakenfor 2 hours at ambient temperature to ensure full adsorption, and thenhistidine buffer components are added (10mM histidine buffer, pH 6.5).

The compound can also be used as an arginine salt monohydrate (obtainedby mixing 98 mg of the compound with 1.7 ml of 0.1 M arginine in 80/20methanol/water to give a 57 mg/ml solution, followed by addition of 7 mlethanol to precipitate the salt) in which case it is seen that the NaOHis not required for solubilisation prior to mixing with the Al—H.

Four different mixtures are prepared, giving a final K2 concentration of10, 50, 250 or 500 μg/ml (to provide a 1, 5, 25 or 50 μg dose of K2 in a100 μl dosage volume); the Al—H concentration is always 3 mg/ml. At allstrengths >95% of compound K2 is adsorbed to the Al—H. The adsorbedadjuvant is referred to hereafter as “Al—H/K2”.

Adjuvant Adsorption to Antigens

3-valent (DTaP) vaccines were adjuvanted with Al—H alone or withAl—H/K2. These formulations showed optimal pH (6.5-6.8±0.1) andosmolarity values (0.300±50 mO). Osmolarity was adjusted with NaCl.Adsorption was detected by separating the adjuvant-antigen complexesfrom unadsorbed antigens by centrifugation. 0.4% DOC was added to thesupernatant containing the unadsorbed antigens. Antigens wereprecipitated by the addition of 60% TCA and collected by centrifugation.The pellet containing the TCA-precipitated antigens was resuspended inloading buffer and loaded onto an SDS-PAGE gel. The pellet containingthe adjuvant-antigen complexes was resuspended in desorption buffer (4×concentration: 0.5 M Na2HPO₄ pH, 8 g SDS, 25 g glycerol, 6.16 g DTT andbromophenol blue), the aluminium hydroxide was removed by centrifugationand the supernatant applied to an SDS-PAGE gel.

Using Al—H alone at a concentration of 2 mg/ml, the adsorption profilesfor DT, TT, PT, FHA and 69K detected by Coomassie Blue staining werecomplete. No bands were detected in the DOC-TCA-treated supernatants.Western Blot analysis confirmed complete Al—H adsorption for DT, TT, PT,FHA and 69K.

Four different K2 concentrations were tested (0.1, 0.025, 0.01, 0.005mg/ml). The Al—H concentration was constant at 2 mg/ml. Even at 0.1mg/ml K2 all antigens were completely adsorbed.

Immunogenicity Testing with TLR Agonists

Four vaccines were tested, each containing (per 0.5 ml) 5 Lf tetanustoxoid, 2 Lf of diphtheria toxoid, and 16 μg acellular pertussisantigens (a mixture of purified PT-9K/129G, FHA and p69 pertactin).

The four vaccines were (A) unadjuvanted (B) adjuvanted with 2 mg/ml Al—H(C) adjuvanted with 2 mg/ml Al—H plus 100 μg/ml synthetic monophosphoryllipid A i.e. a TLR4 agonist, or (D) adjuvanted with 2 mg/ml Al—H plus 1mg/ml compound ‘K2’ i.e. a TLR7 agonist. The TLR agonists in vaccines(C) and (D) were adsorbed to the Al—H. All antigens were adsorbed to theAl—H in formulations (B), (C) and (D).

For comparison the BOOSTRIX™ product was also tested. As discussed aboveit contains (per 0.5 ml) 2.5 Lf of diphtheria toxoid, 5 Lf tetanustoxoid, and 18.5 μg acellular pertussis antigens (a mixture of purifiedPT, FHA and p69 pertactin), and it is adjuvanted with a mixture ofaluminium phosphate and hydroxide salts. A mixture of buffer and Al—Hwas used as a negative control.

The four vaccines were administered to female Balb/C mice (6 weeks old)at 100 μl intramuscular doses on days 0, 21 and 35. Sera were tested 2weeks after each dose.

Serum total IgG titers were measured against each antigen and were asfollows (geometric means):

Day Ag Unadj Al—H Al—H + K2 Al—H + MPL Boostrix −ve control 14 Dt 0.0300.603 8.119 1.762 1.205 0.030 Tt 0.191 2.546 46.14 14.49 3.217 0.030 PT14.28 10.26 26.68 16.61 4.685 1.080 FHA 0.145 0.579 27.38 7.942 1.4580.126 p69 0.566 7.046 72.47 38.49 12.39 0.129 35 Dt 0.030 69.29 490.1139.3 89.59 0.030 Tt 48.98 128.6 808.9 298.0 109.8 0.030 PT 245.0 267.5377.1 695.9 195.0 2.734 FHA 7.847 65.25 400.6 231.0 49.49 0.030 p6932.50 222.2 1484 575.8 318.0 0.050 49 Dt 0.055 79.74 452.9 149.2 88.950.047 Tt 60.59 123.2 694.3 317.0 105.6 0.030 PT 200.8 387.7 329.7 642.3300.0 2.065 FHA 20.96 72.10 462.4 272.5 78.98 0.030 p69 107.6 384.9 1275794.5 302.6 0.134

Thus in all cases and at all time points (except for PT at day 49) thehighest titers in these 6 groups were seen in the mice which hadreceived the antigens adjuvanted with adsorbed TLR agonist, and theaddition of a TLR agonist to Al—H improved IgG responses relative toAl—H alone. Importantly, improved responses were seen in all cases whencompared to the licensed BOOSTRIX™ vaccine. Moreover, unlike Al—H aloneor BOOSTRIX™, the adsorbed TLR agonists were consistently able toimprove anti-PT titers relative to the unadjuvanted group.

The use of the TLR agonists also leads to more rapid responses. Thesecond dose showed a clear increase in IgG responses for all antigens,but the improvements after the third dose were not so significant. Themice in these experiments were naive to DTP but in a real-world humansituation the target patients will previously have received DTP vaccineas a child and so the rapid response seen after the second dose in theseexperiments is helpful.

Immunogenicity Testing with Oil-in-Water Emulsion

Vaccines were prepared containing (per 0.5 ml) 5 Lf tetanus toxoid, 2 Lfof diphtheria toxoid, and 16 μg acellular pertussis antigens (a mixtureof purified PT-9K/129G, FHA and p69 pertactin). These were administeredto female Balb/C mice (6 weeks old) at 100 μl intramuscular doses ondays 0, 21 and 35. Sera were tested 2 weeks after each dose. Vaccineswere (A) unadjuvanted or (B) adjuvanted with MF59 emulsion, by mixing 50μl antigen solution with 50 μl MF59. For comparison the BOOSTRIX™product was also tested, and an antigen-free negative control was alsotested.

Serum total IgG titers were measured against each antigen and were asfollows (geometric means):

Day Ag Unadj MF59 Boostrix −ve control 14 Dt 0.030 0.038 1.308 0.030 Tt0.064 3.025 2.480 0.030 PT 7.255 9.448 1.629 1.517 FHA 0.034 0.026 0.8440.137 p69 1.365 9.281 14.46 1.670 35 Dt 0.030 115.2 85.55 0.033 Tt 24.31379.6 83.61 0.060 PT 247.4 391.9 112.7 3.582 FHA 7.433 148.8 37.58 0.068p69 11.44 1006 398.3 3.817 49 Dt 0.5165 78.54 92.18 0.054 Tt 40.21 337.599.09 0.030 PT 353.6 480.8 162.8 5.231 FHA 13.32 218.1 65.34 0.119 p6936.91 1036 403.6 1.952

Thus after two doses in DTP-naive mice the emulsion-adjuvanted vaccinegave much better antibody titers than the approved BOOSTRIX™ product.This superiority was maintained after a third dose, except for theanti-Dt response. Moreover, unlike BOOSTRIX™, the emulsion was able toimprove anti-PT titers relative to the unadjuvanted group.

FHA-Specific Memory B Cells

Four-to-five months after the third dose, FHA-specific memory B cellswere measured in the immunised mice. The mice were sacrificed and theirspleen cells were cultured in the presence of IL-2 and CpG for 5 days inorder to expand all memory B cells. Spleen cells were then harvested andseeded in 96-well ELISPOT plates previously coated with either FHAantigen (10 mg/ml) or anti-mouse Ig. After overnight incubation, plateswere washed to remove unattached spleen cells and both FHA-specific andtotal memory B cells were detected by biotinylated anti-mouse Ig andHRP-streptavidin. Colored spots, representing individual memory B cell,were counted with an ELISPOT reader instrument. The percentage ofFHA-specific B cells compared to total B cells was then calculated foreach sample, and FIG. 1 shows the results for the following groups: (A)unadjuvanted; (B) adjuvanted with Al—H; (C) adjuvanted with Al—H plusMPL-A; (D) adjuvanted with Al—H plus ‘K2’; (E) adjuvanted with MF59; (F)BOOSTRIX™; and (G) negative control. The highest response was seen ingroup (D) i.e. using the adsorbed TLR7 agonist. The next highestresponses were seen in groups (C) and (E), with either the adsorbed TLR4agonist or the emulsion, where responses were essentially the same butwere still higher than with the BOOSTRIX™ product.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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1. An immunogenic composition comprising a diphtheria toxoid, a tetanustoxoid, a pertussis toxoid, an aluminium salt adjuvant, and a TLRagonist, wherein the composition comprises an excess (measured in Lfunits) of tetanus toxoid relative to diphtheria toxoid.
 2. Thecomposition of claim 1, wherein the TLR agonist is a TLR4 agonist or aTLR 7 agonist.
 3. The composition of claim 1, wherein the TLR agonist isadsorbed to the aluminium salt adjuvant.
 4. The composition of claim 1,wherein 1, 2 or 3 of the toxoids is/are adsorbed to the aluminium saltadjuvant.
 5. The composition of claim 1, with an Al⁺⁺⁺ concentration≤0.5 mg/ml.
 6. The composition of claim 1, wherein the aluminium salt isan aluminium hydroxide.
 7. The composition of claim 1, wherein the TLRagonist is a compound of formula (K), or a pharmaceutically acceptablesalt thereof.
 8. The composition of claim 1, wherein the TLR agonist iscompound K2.
 9. The composition of claim 1, wherein the TLR agonistcomprises at least one adsorptive moiety which allows it to adsorb toinsoluble metal salts.
 10. The composition of claim 9, wherein theadsorptive moieties is a phosphate or a phosphonate.
 11. The compositionof claim 1, wherein the TLR agonist has formula (C), (D), (E), (F), (G),(H), (I), (II), (J) or (K) as defined in the description, or is a3d-MPL.
 12. The composition of claim 1, wherein the TLR agonist is oneof compounds 1 to 102 as defined in reference 13, or a pharmaceuticallyacceptable salt thereof.
 13. The composition of claim 1, furthercomprising a histidine buffer.
 14. The composition of claim 1, with adiphtheria toxoid concentration ≤4 Lf/ml.
 15. The composition of claim1, with a tetanus toxoid concentration ≤9 Lf/ml.
 16. The composition ofclaim 1, with a pertussis toxoid concentration ≤4 μg/ml.
 17. Thecomposition of claim 1, further comprising a trivalent inactivatedpoliovirus antigen component.
 18. The composition of claim 1, having apH between 6.1 and 7.9.
 19. A method of raising an immune response in asubject, comprising the step of administering to the subject thecomposition of claim
 1. 20. The method of claim 19, wherein the subjecthas previously received a DTP vaccine as a child.
 21. An immunogeniccomposition comprising a diphtheria toxoid, a tetanus toxoid, apertussis toxoid, and an oil-in-water emulsion adjuvant, wherein thecomposition comprises an excess (measured in Lf units) of tetanus toxoidrelative to diphtheria toxoid.
 22. The composition of claim 21, whereinthe emulsion comprises squalene and/or polysorbate
 80. 23. Thecomposition of claim 21, wherein at least 80% by number of oil dropletsin the emulsion have a diameter of less than 220 nm.
 24. The compositionof claim 21, with a diphtheria toxoid concentration ≤4 Lf/ml.
 25. Thecomposition of claim 21, with a tetanus toxoid concentration ≤9 Lf/ml.26. The composition of claim 21, with a pertussis toxoid concentration≤4 μg/ml.
 27. The composition of claim 21, further comprising atrivalent inactivated poliovirus antigen component.
 28. The compositionof claim 21, having a pH between 6.1 and 7.9.
 29. A method of raising animmune response in a subject, comprising the step of administering tothe subject the composition of claim
 21. 30. The method of claim 29,wherein the subject has previously received a DTP vaccine as a child.31. An immunogenic composition comprising an acellular pertussiscomponent in which a pertussis toxoid, filamentous hemagglutinin andpertactin are present at a mass ratio of 1:1:2.
 32. The composition ofclaim 31, wherein the pertussis toxoid is the PT-9K/129G double mutant.33. The composition of claim 31, in unit dose form, and comprising 4 μgpertussis toxoid, 41g FHA and 8 μg pertactin per unit dose.
 34. Thecomposition of claim 31, further comprising diphtheria toxoid andtetanus toxoid.
 35. The composition of claim 31, further comprising analuminium salt adjuvant.
 36. The composition of claim 35, comprising aTLR agonist adsorbed to the aluminium salt.